Among all described serotonin (5-HT) receptors in mammals, the type three (5-HT3) is the only ligand-gated ion channel receptor for serotonin. By using double in situ hybridization histochemistry, we found co-expression of the functional 5-HT3A subunit of the 5-HT3 receptor and the central CB1 cannabinoid receptor in neurons of the rat telencephalon. Double-labeled 5-HT3A/CB1 neurons were found in anterior olfactory nucleus, superficial and deep layers of cortex, hippocampal formation (hippocampus, dentate gyrus, subiculum and entorhinal cortex) and amygdala. Analysis of the proportion of neurons co-expressing 5-HT3A and CB1 receptors in cortex and amygdala showed that depending on the brain region, 37-53% of all neurons expressing the 5-HT3A subunit also express CB1 transcripts; while 16-72% of the total population of neurons expressing CB1 mRNA co-express the 5-HT3A subunit. By using a combination of double in situ hybridization and immunohistochemistry, we demonstrated that 5-HT3A/CB1-expressing neurons contained the inhibitory neurotransmitter g-amino butyric acid (GABA). These results imply that in distinct regions of the telencephalon, GABA neurons that react to cannabinoids may also be responsive to serotonin through 5-HT3 receptors. However, to determine possible overlap of CB1 and 5-HT3 receptors at the subcellular level, it would be necessary to investigate the ultrastructural distribution of 5-HT3 receptors in relation to the subcellular compartmentalization of CB1 receptors. Thus, we are using peptides and fusion proteins as antigens to develop polyclonal antibodies against the 5-HT3A and CB1 proteins. The specificity of these antibodies will be evaluated by their selective recognition of the antigenic peptide and receptor protein (obtain by in vitro translation of mRNA), by their selective pattern of immuno-staining on the brain and immuno-staining of same neurons that express the corresponding mRNA. Cellular co-existence of 5-HT3A and CB1 transcripts in interneurons of cortex, hippocampal formation and amygdala suggest possible interactions between the cannabinoid and serotonergic systems at the level of GABA neurotransmission in brain areas involved in cognition, memory and emotion.